Transferring exogenous genetic material into cells is the basis for modern molecular biology. The continuing development of novel methods for improving the efficiency, specificity, and/or size limitations of the transfer process has broadened the scope of research and product development by enabling the production of polynucleotide clones and recombinant organisms that previously were impractical or impossible to construct. Calcium phosphate precipitation, electroporation, lipofection, ballistic transfer, DEAE-dextran transfection, microinjection, and viral-based transfer methods, among others, have been described for introducing foreign DNA fragments into mammalian cells.
The art also has developed yeast artificial chromosome ("YAC") cloning vectors which are capable of propagating large (50 to more than 1000 kilobases) cloned inserts (U.S. Pat. No. 4,889,806) of xenogenic DNA. YAC clone libraries have been used to identify, map, and propagate large fragments of mammalian genomic DNA. YAC cloning is especially useful for isolating intact genes, particularly large genes having exons spanning several tens of kilobases or more, and genes having distal regulatory elements located tens of kilobases or more upstream or downstream from the exonic sequences. YAC cloning is particularly advantageous for isolating large complex gene loci, such as unrearranged immunoglobulin gene loci, and genes which have been inexactly mapped to an approximate chromosomal region (e.g., a Huntington's chorea gene). YAC cloning is also well-suited for making vectors for performing targeted homologous recombination in mammalian cells, since YACs allow the cloning of large contiguous sequences useful as recombinogenic homology regions in homologous targeting vectors. Moreover, YACs afford a system for doing targeted homologous recombination in a yeast host cell to create novel, large transgenes (e.g., large minigenes, tandem gene arrays, etc.) in YAC constructs which could then be transferred to mammalian host cells.
Unfortunately, manipulation of large polynucleotides is problematic. Large polynucleotides are susceptible to breakage by shearing forces and form highly viscous solutions even at relatively dilute concentrations, making in vitro manipulation exceedingly difficult. For these reasons, and others, it is desirable to reduce the amount of manipulation that YAC clones and other large DNA fragments are subjected to in the process of constructing large transgene constructs or homologous recombination constructs.
More problematic is the fact that the transfer of large, intact polynucleotides into mammalian cells is typically inefficient or provides a restriction on the size of the polynucleotide transferred. For example, Schedl et al. (1992) Nucleic Acids Res. 20: 3073, describe transferring a 35 kilobase YAC clone into the mouse genome by pronuclear injection of murine embryos; however, the shear forces produced in the injection micropipette will almost certainly preclude the efficient transfer of significantly larger YAC clones in an intact form. Many large genes likely could not be transferred efficiently into mammalian cells by current microinjection methods.
Spheroplast fusion has been used to introduce YAC DNA into fibroblasts, embryonal carcinoma cells, and CHO cells (Pachnie et al. (1990) Proc. Natl. Acad. Sci. (U.S.A.) 87: 5109; Payan et al. (1990) Mol. Cell. Biol. 10: 4163; Chirke et al. (1991) EMBO J. 10: 1629; Davies et al. (1992) Nucleic Acids Res. 20: 2693). Alternative transfection methods such as calcium phosphate precipitation and lipofection have been used to transfer YAC DNA into mammalian cells (Eticciri et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 2179; Strauss W and Jaenisch R (1992) EMBO J. 11: 417).
Thus, there exists a need in the art for an efficient method for transferring large segments of DNA, such as large YAC clones, into mammalian cells, such as embryonic stem cells for making transgenic animals, with a minimum of manipulation and cloning procedures. In particular, it would be highly advantageous if it were possible to isolate a large cloned mammalian genomic fragment from a YAC library, either linked to YAC yeast sequences or purified away from YAC yeast sequences, and transfer it intact into a mammalian host cell (e.g., an ES cell) with a second polynucleotide sequence (e.g., a selectable marker such as a neo.sup.r expression cassette) without additional cloning or manipulation (e.g., ligation of the sequences to each other). Such a method would allow the efficient construction of transgenic cells, transgenic animals, and homologously targeted cells and animals. These transgenic/homologously targeted cells and animals could provide useful models of, for example, human genetic diseases such as Huntington's chorea and Alzheimer's disease, among others.